Project summary/abstract The overall goal of this project is to develop and validate a novel type of sensor that allows near-infrared (NIR) photo-controllable ratiometric imaging of Fe2+ and Fe3+ in Alzheimer?s disease (AD) animal models simultaneously with high spatial and temporal resolution and to understand the role of iron redox equilibrium in ferroptosis-related AD. The central hypothesis is that, in addition to the role of changes in total iron content in AD that many researchers have been focusing on, the iron redox equilibrium may also play a critical role in ferroptosis-dependent AD progression. AD is an incurable neurodegenerative disease which affects millions of people around the world. In recent years, accumulating evidence points to ferroptosis, a newly discovered type of programmed cell death depends on iron biology and iron redox balance, as a possible cause of neuron cell death associated with AD. Labile iron, which is the pool of iron not bound to proteins or other biomolecules, is involved in lipid peroxidation and can directly lead to ferroptosis through Fenton-mediated lipid peroxidation. Aberrant high levels of iron and lipid peroxidation were also detected in AD brains, indicating a relationship between labile iron, ferroptosis, and AD. Fe2+ and Fe3+ can both trigger lipid peroxidation separately. However, the ratio of Fe2+/Fe3+ or iron redox equilibrium significantly increases the speed of fatal lipid hydroperoxide accumulation. Despite the importance of such iron redox equilibrium, the precise mechanism of iron redox activity in AD remains to be understood. A major barrier in addressing this hypothesis is the lack of current methods for simultaneous imaging of Fe2+ and Fe3+ selectively in vivo with high spatial and temporal resolution. To overcome this technical barrier and to achieve the above overall goal, we propose to build upon the methods developed in the current R01 grant and to further develop them into DNAzyme sensors for investigation of iron redox equilibrium in AD in three aims: 1) Transform the current sensor design from UV-controlled DNAzyme fluorescent sensors that can identify Fe2+ and Fe3+ qualitatively into NIR photo-controlled fluorescent imaging sensors for simultaneous quantification of Fe2+/Fe3+ in cells using fluorescence resonance energy transfer (FRET); 2) Use the fluorescent imaging (FI) sensors to investigate the roles of Fe2+ and Fe3+ and their redox equilibrium in live zebrafish and brain sections of AD mouse; and 3) Upgrade the FI sensors into photoacoustic imaging (PAI) sensors (with NIR photo-control) for deep-tissue imaging of Fe2+ and Fe3+ and its redox equilibrium in zebrafish and brain sections of AD mice. Through the further development of our current FI sensors and by enhancing them to act as PAI sensors, this supplement will provide spatiotemporal information of iron in different oxidation states simultaneously in live zebrafish and in brain sections of mouse AD models, and thus advance the AD field by providing an essential tool needed to gain deeper insight into the role of redox activities and homeostasis of iron in AD.